Alkaline phosphatase (EC 3.1.3.1.) (abbreviation: ALP) is a metalloenzyme consisting of a group of isoenzymes present in different tissues of animal organisms and in particular in man.
Alkaline phosphatase isoenzymes are important in protocols for diagnosing different conditions in adults or in children, and a number of methods have been proposed for separating and assaying such isoenzymes. ALP isoenzymes can be divided into four classes: non-specific tissue (bone, liver and kidney), adult intestinal, foetal intestinal, and placental. A number of variations can exist within a single class, namely:
hepatic: hepatic 1 (H1), hepatic 2 (H2);
ultrafast (UF);
osseous (Os);
placental: placental 1 (P1), placental 2 (P2);
intestinal: intestinal 1 (I1), intestinal 2 (I2), intestinal 3 (I3).
Thus nine principal fractions can be distinguished which have to be separated, identified and quantified in particular for their detection into hepatic and biliary disorders and into certain bone diseases, including osseous tumours or Paget""s disease.
Reference will occasionally be made below to the term xe2x80x9cfractionxe2x80x9d to designate a class of ALP isoenzymes or a particular variant within a class of isoenzymes. On the electrophoresis support, a xe2x80x9cfractionxe2x80x9d corresponds to a band revealed after migration.
The most frequent routine analysis carried out on alkaline phosphatase isoenzymes consists of measuring the total enzymatic activity using a substrate of this enzyme, generally para-nitrophenylphosphate. That method, however, cannot determine the levels of the different isoenzymes.
The principal method for separative analysis of such compounds uses electrophoretic techniques. Isoelectrofocussing is occasionally used and can separate 10 to 20 bands depending on the procedure used. Identifying all of the bands is difficult, rendering clinical interpretation extremely awkward.
Zone electrophoresis enables a good separation of the principal forms of the isophosphatases. However, certain fractions are superimposed, in particular the Os, H1 and P1 fractions, and thus complementary treatments have to be carried out to separate and identify them. Such treatments must be carried out on the biological test samples to be tested before depositing them onto the gel.
Such treatments consist, for example, of thermal denaturing, incubation with specific inhibitors such as urea, amino acids, etc., enzymatic incubation with neuraminidase, ficin, phospholipase C, incubation with specific antiplacental or anti-intestinal antisera.
Several separation procedures which are in current use have been dealt with by Van Hoof V. O., De Broe Marc, E., Clinical Laboratory Sciences, vol. 31, issue 3 1994, xe2x80x9cInterpretation and clinical significance of alkaline phosphatase isoenzyme patternsxe2x80x9d.
One particular procedure has been proposed in United States patent U.S. Pat. No. 5,264,098 which describes the separation of ALP isoenzymes using a gel electrophoresis reaction employing a gel buffer containing at least one non ionic detergent and an anionic detergent.
Available treatments for identifying and quantifying ALP isoenzymes have certain disadvantages as regards routine analysis. In addition to high costs, they can on the one hand be long and can considerably complicate manipulation, and on the other hand, complete determination (of all of the isoenzymes) necessitates a plurality of treatments (2 or 3) for a single sample, limiting the number of samples which can be simultaneously analysed on the one gel.
Other treatments have been proposed which, for example, recommend treating the sample prior to loading onto the electrophoresis gel. In this regards, the action of the WGA lectin (wheat germ agglutinin) is particularly interesting (see Sidney B. Rosalki, A. Ying Foo, Clinical Chemistry, 30/7, p. 1182-1186, 1984, xe2x80x9cTwo methods for separating and quantifying bone and liver alkaline phosphatase isoenzyme in plasmaxe2x80x9d, European patent EP-A-0 131 606 dated 5/11/86). EP-A-0 131 606 describes the differential detection of bone and liver ALP isoenzyme comprising treating the test sample with lectin, then incubating the mixture obtained followed by separating the ALP bound to the lectin from the fraction containing free ALP and determining the ALP activity in one of the two media or in both. In a particular implementation of that patent, the two fractions (ALP bound to lectin and free ALP) are separated by electrophoresis.
With the exception of the intestinal forms, all isophosphatases possess sialic acids and are thus affected by a treatment with WGA lectin to a greater or lesser extent. The osseous fraction is the most sialated and thus is affected the most by this treatment, which under suitable conditions retards its mobility and thus causes it to precipitate in a zone which is distinct from the zone where the hepatic fraction is located.
In order to render ALP isoenzyme precipitation more selective towards the osseous isoenzyme, certain authors have used detergents such as Triton X100 (Rosalki). However, despite the presence of such detergents, residual interactions of the WGA lectin with other isophosphatases subsist, which cause co-precipitation of such fractions with the osseous fraction.
In addition to this lack of specificity, a further disadvantage of this technique is to render the analysis considerably more complicated.
The publication by Rosalki S. B. et al, cited above, alternatively proposes incorporating lectin into the buffer used to impregnate the electrophoresis gel prior to using this gel. This dispenses with prior treatment of the sample. In that case, the majority of the osseous fraction is precipitated close to where the sample has been loaded. The mobility of all of the other isoenzymes with the exception of intestinal isoenzymes is affected by the action of the WGA lectin despite the presence of the detergents mentioned above.
In the context of that treatment, the properties of the lectin used are its ability to interact specifically with the ALP isoenzymes which contain sialic acids.
The present invention proposes means for at least partially overcoming the disadvantages stated in prior art methods. In particular, the invention defines a method enabling separation and identification of ALP isoenzymes which is improved as regards specificity and sensitivity.
The present invention also provides consumers, in particular clinicians, with a process for separating, identifying and quantifying the principal alkaline isophosphatases, which process can be carried out in a single step on a single electrophoresis support which is easy to produce.
The invention thus proposes a novel process for separating and identifying ALP isoenzymes by electrophoresis, characterized in that the lectin is deposited on the electrophoresis support in a localised manner.
The lectin deposited in solution in a localised manner can diffuse into the support while remaining localised in a determined zone of this support during electrophoretic migration.
The deposit in question, located close to the zone where the sample is deposited, is distinguished from the uniform loading over an extended zone or over the whole of the electrophoresis support as described in the prior art.
The invention thus provides a process for separating alkaline phosphatase isoenzymes from a biological sample by electrophoresis, characterized in that the electrophoresis reaction is carried out on an electrophoresis support after depositing a solution of lectin onto the electrophoresis support in a given zone, under conditions which permit interaction between said lectin and the ALP isoenzymes contained in the analysed biological sample, deposition of the lectin solution further being carried out under conditions which are suitable to allow separation of the ALP isoenzymes constituted by the osseous fraction and by the hepatic fraction.
The interaction in question leads to the formation of a complex between the lectin and the ALP isoenzyme until equilibrium is obtained.
The process of the invention enables the osseous fraction of the ALP to be acted on in a manner which is more specific than on the other ALP fractions because of the reaction of this fraction with the lectin.
The biological sample analysed can be any biological sample which may contain ALP, in particular a biological fluid sample such as a serum or plasma sample, or possibly a tissue sample removed from a patient.
In the invention, electrophoresis is carried out on any suitable electrophoresis support, in particular on a gel, more particularly on an agarose or a polyacrylamide gel, or on a porous membrane, in particular made of cellulose acetate.
The particular conditions defined above for carrying out the electrophoresis of the invention can be applied in the context of known electrophoresis methods which may or may not be automated.
The localised lectin deposit zone is determined as a function of the direction of migration of the sample and the lectin. Thus the lectin deposit zone is selected such that, during migration, the sample traverses the lectin, the mobility of the latter during migration having been taken into consideration. Similarly, the final position normally reached by the other ALP isoenzymes is taken into consideration in order to determine the lectin deposit zone with respect to that of the sample. In practice, the lectin and the sample are 1 to 10 mm apart, advantageously 5 mm, when the sample is loaded.
The other conditions for localised depositing of the lectin, such as the concentration of the lectin, the time for application to the electrophoresis support, are determined such that they enable osseous and hepatic ALP isoenzymes to be separated during electrophoresis under the migration reaction conditions.
In other words, once the parameters of electrophoresis have been determined particularly as regards depositing the lectin, the process of the invention can separate the osseous and hepatic isoenzymes under conditions which are satisfactory for identifying them with respect to the other ALP isoenzymes, and preferably to quantify them. The mobility of the other ALP isoenzymes, affected during passage through the zone where the lectin is present, returns to normal outside this zone.
Since the osseous ALP fraction is more sialated, its electrophoretic migration is the most affected by the passage of the sample through the lectin deposited on the support. As a result it is precipitated in a zone which may be distinguished from the migration zone of other ALP isoenzymes.
In a preferred embodiment of the invention, a process for separating alkaline phosphatase (ALP) isoenzymes by electrophoresis is characterized in that it comprises:
depositing the biological sample containing the ALP isoenzymes to be separated on the electrophoresis support;
depositing a lectin solution which can interact with the ALP isoenzymes contained in the sample on the electrophoresis support;
applying an electric field to permit electrophoretic separation by migration of the ALP isoenzymes under conditions which can permit differential separation of the osseous and hepatic ALP fractions;
revealing the separated ALP isoenzymes.
Revealing is carried out using any known means, preferably using an ALP substrate.
At the end of the steps resulting in separation of the ALP isoenzymes on the electrophoresis support, a quantitative analysis of the different separated isoenzymes, or certain of them, can be carried out.
Different methods can be used to quantify the ALP isoenzymes detected at the end of electrophoresis. As an example, the densitometry of the electrophoresis support can be measured after staining the separated ALP fractions using an ALP substrate.
When the osseous ALP fraction is in excess with respect to the fixing capacity of the lectin deposited on the gel, the lectin may not precipitate the whole of this fraction. In this case, the non precipitated portion continues to migrate with the other fractions, and can be found in the form of a smear close to the subjacent isoenzymes, in particular H2, I1, I2 and I3. In this case, the different isoenzymes can be quantified by depositing the same sample a second time on the same support, in the absence of lectin. On the xe2x80x9cno lectinxe2x80x9d pattern, the percentages of the H1 Os P1 block and the separated fractions H2, I1, P2, I2, I3 and UF are determined. The percentage of the Os fraction is obtained by deducting the percentages of Hi and Pi determined from the xe2x80x9cwith lectinxe2x80x9d profile from the H1 Os P1 block.
Within the context of the definitions given above, when carrying out electrophoresis in the presence of a localised deposit of lectin on the electrophoresis support, the lectin and the sample can be deposited simultaneously on the electrophoresis support.
In another embodiment of the invention, the sample and the lectin are deposited at different times.
Further, the sample and the lectin solution can respectively be deposited on the electrophoresis support over periods which may be identical or different.
Advantageously, within the context of the definitions given above, if necessary taken in combination, the lectin and the sample are deposited over a substantially identical period of time, and preferably simultaneously for practical and economic reasons.
For a given sample and a determined concentration of lectin in the solution, the sample application and lectin application period, and thus the quantity of sample and lectin deposited on the electrophoresis support, is selected so as to precipitate the Os fraction of the ALP isoenzymes such that the osseous and hepatic fractions are separated during migration. To determine the concentration of the lectin solution and the period over which the solution is applied to the electrophoresis support, the temperature reached by the support during migration is also taken into account.
Indeed, since the interaction of the lectin with the osseous ALP fraction is dependent on temperature, the temperature of the electrophoresis support during the migration step must be taken into account. A reduction in the lectin-osseous ALP interaction due to an increase in the temperature of the electrophoresis support can be compensated for by an increase in the quantity of lectin deposited, for example by increasing the concentration of the lectin solution used.
These parameters can be determined in the light of the indications below and the values given in the examples and can if necessary be adapted to the selected electrophoresis conditions, by carrying out tests such as those which are given in the examples below.
The sample application and/or lectin solution application period can vary and can in particular be in the range of 5 to 20 min, preferably 15 minutes, for the sample and/or for the lectin.
Depositing can be carried out using any known manual or automated means, for example using xe2x80x9ccombxe2x80x9d type applicators, for example the devices described in European patent application EP-A-0 493 996.
The lectin used is in the form of an aqueous solution.
Thus the concentration of lectin deposited on the electrophoresis support used under normal conditions is in the range of 0.1 mg/ml to the limit of solubility of lectin in water. This concentration is advantageously in the range of 0.5 to 15 mg/ml, preferably in the range of 1 to 10 mg/ml.
In a particular implementation of the invention, the process is characterized in that when the lectin concentration is in the range of 1 to 10 mg/ml, the migration temperature is respectively in the range of 18xc2x0 C. to 53xc2x0 C. These conditions are applicable inter alia to depositing a lectin solution over a period of close to about 15 minutes with a comb type applicator as described in European patent application EP-A-0 493 996.
The invention particularly concerns an implementation of an electrophoresis process in which, when the ALP isoenzymes present in the test sample have an electrophoretic mobility in the direction of the anode, the lectin is deposited between the anode and the sample deposit zone.
Advantageously in this case, the lectin is deposited between the biological sample deposit point and the zone normally occupied by intestinal fraction 3 (I3) at the end of the migration step, when this fraction is present in the sample.
Different lectins can be used in the invention. Lectins are proteins which fix sialated groups. Lectins which can be used in the present invention which can be cited include wheat germ (Triticum vulgaris) lectin, or WGA (Wheat Germ Agglutinin). WGA lectin can be obtained from Sigma, Pharmacia, etc.
The invention also concerns a process satisfying the definitions given above, taken separately or in combination, in which separation of fractions other than the osseous and hepatic 1 of ALP is improved.
The invention thus makes available, under conditions which are compatible with routine laboratory analyses, means for in vitro detection in a biological sample of the abnormal presence of one or more ALP isoenzymes.
This process can in particular be carried out during a protocol for diagnosing a hepatic or biliary disorder corresponding to the abnormal presence of the hepatic ALP fraction. The invention can also enable the abnormal presence of the osseous fraction to be researched in the context of the diagnosis of osseous disorders.
The invention also concerns kits for carrying out separation of fractions constituted by ALP isoenzymes by electrophoresis.
The process of the invention thus has the advantage of using a single electrophoresis support, without prior treatment of the sample, to carry out qualitative determination of all of the ALP isoenzymes in one deposit and their quantitative determination in two deposits onto a single support. The electrophoresis support is free of lectin prior to its use, which enables it to be stored under the usual temperature conditions and does not change its manufacturing cost.
A kit comprises, for example:
an electrophoresis support comprising a porous material suitable for depositing a biological sample to be analysed and for carrying out electrophoretic migration;
a solution of lectin in a concentration in the range of 0.1 mg/ml to 15 mg/ml, advantageously in the range of 1 to 10 mg/ml, preferably in the range of 1 to 10 mg/ml.
The kit of the invention can also comprise the buffer or buffers required for the electrophoresis reaction. It can also contain ALP activity revealing reagents.
Further advantages and features of the invention will become more clear from the following examples.